Established tumors create a condition of immunologic unresponsiveness (tolerance) toward their own antigens. This constitutes a barrier to the clinical immunotherapy of cancer, and represents a striking model of acquired peripheral tolerance. This project will address the hypothesis that murine plasmacytoid dendritic cells (pDCs) expressing the tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO) are selectively recruited to tumor-draining lymph nodes, where they create tolerance to tumor antigens. Aim 1 will test the hypothesis that IDO-expressing DCs from tumor-draining lymph nodes directly suppress T cell responses to tumor-derived antigens. This will first be done by adoptively transferring DCs isolated from murine tumor-draining lymph nodes, to test the hypothesis that these DCs render defined antigen-specific CD8+ T cells anergic in vivo, via IDO-induced tryptophan-withdrawal stress and cell-cycle arrest. Then a B16-OVA tumor model will be used with defined OVA-specific T cells to test the hypothesis that IDO expression in by pDCs in tumor-draining lymph nodes creates anergy to tumor-derived antigens in vivo. Aim 2 will test the hypothesis that IDO-expressing DCs from tumor-draining LNs amplify and sustain their effect by creating tumor-specific regulatory T cells. First, an in vitro model will be used to test the hypothesis that pDCs from tumor-draining lymph nodes suppress normal cell-cycle progression and effector-cell differentiation of CD4+ T cells via expression of IDO, and that this drives the conversion of naive CD4+ T cells into antigen-specific Tregs. Then an in vivo tumor model and defined CD4+ T cells will be used to test the hypothesis that antigen-presentation by IDO+ cells in vivo creates new antigen-specific Tregs against a nominal tumor-derived antigen. Finally, genetically defined mice will be used to test the hypothesis that pDCs in tumor-draining lymph nodes constitutively express IDO because they receive CTLA4->B7 signals delivered by tumor-specific Tregs. Taken together, the proposed studies will provide mechanistic insight into the role of IDO in the pathologic state of tolerance displayed toward tumors. This is relevant to the basic biology of tumors and to acquired peripheral tolerance, and has direct implications for clinical immunotherapy of cancer.